Journal of Lipid Research, Vol. 21, 316-325, March 1980
Copyright © 1980 by Lipid Research, Inc.

Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography

Karl H. Weisgraber and Robert W. Mahley

Gladstone Foundation Laboratories for Cardiovascular Disease, University of California, San Francisco, CA 94140 and National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205

A reproducible and quantitative subfractionation of human high density lipoproteins (HDL) by heparin-Sepharose affinity chromatography has been developed. Two elution methods (A and B) were used to subfractionate HDL₂ (d 1.063-1.125 g/ml) or total HDL (d 1.063-1.21 g/ml). Method A separated HDL₂ into three subclasses, each with distinct chemical properties and in vitro metabolic characteristics. The first subclass, referred to as HDL₂-without E, passed through the affinity column unretarded and represented approximately 85% of the HDL₂ lipoprotein protein. HDL₂-without E contained the A-I, A-II, and C apoproteins which characterize typical HDL. The second subclass eluted from the column (7-10% of the protein) contained, in addition to the A-I and A-II apoproteins, the E and (E—A-II) apoproteins, and was designated as HDL₂-with E. The B apoprotein was the major protein component of the third subclass eluted from the column (ßbeta; lipoproteins). The ßbeta; subclass accounted for approximately 3-8% of the HDL₂ protein and was similar to Lp(a) in composition and size. Method B further subdivided the ßbeta; subclass into two fractions (ßbeta;₁ and ßbeta;₂) with slightly different electrophoretic mobilities. The various heparin-Sepharose subclasses were further characterized by their ability to compete with ¹²⁵I-labeled low density lipoproteins (LDL) for binding to cell surface receptor sites of fibroblasts. By virtue of the presence of the E apoprotein, HDL₂-with E competed effectively with ¹²⁵I-labeled LDL for binding to the cell surface receptors, whereas HDL₂-without E were ineffective in competing with LDL. The ßbeta; subclass possessed binding capability similar to that of LDL. Subfractionation of HDL by heparin-Sepharose affinity column chromatography provides an attractive alternative to methods based solely on ultracentrifugation, in that it subfractionates HDL into subclasses with differing apoprotein contents that impart distinct metabolic characteristics to each class.—Weisgraber, K. H., and R. W. Mahley. Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography.

Supplementary key words apoprotein E • HDL subclasses • apo(E—A-II) complex • Lp(a) • lipoprotein cell receptors

Submitted on August 29, 1979
Revised on November 20, 1979

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